Long-term combined application of chemical fertilizers and organic manure shapes the gut microbial diversity and functional community structures of earthworms

Earthworms are crucial regulators of many soil ecosystem processes; however, the effects of fertilization regimes on earthworm gut microbiota remains poorly understood. Here, we investigated the effects of 10 years of fertilization on the gut microbiota of two different earthworm species, Metaphire californica and Amynthas phaselus, belonging to anecic and endogeic ecological groups, respectively. Five fertilization treatments were designed as follows: non-nitrogen fertilizer (CK), nitrogen fertilizer only (NPK), and the combined application of inorganic fertilizers and three different application rates of pig manure (NPKM1, NPKM2, NPKM3). We observed that, compared with the CK and NPK treatments, the organic manure treatments had strong effects on both earthworm gut microbial communities and the associated metabolic functions. The lower application rate of organic manure significantly increased the gut microbial diversity of both earthworm species, which may have further improved their gut microbial metabolic potential, especially for the metabolism of various carbon resources. With the increasing application of organic fertilizers, the abundance of several bacterial families, such as Comamonadaceae, Oxalobacteraceae, and Cytophagaceae, significantly increased in the gut of M. californica. These families have also been identified as the primary indicator taxa of high organic matter inputs and play a crucial role in regulating microbial community functionalities. However, a similar trend was not observed in the gut of A. phaselus, indicating that A. phaselus might be less sensitive to fertilization regimes because it lives deeper in the soil and has a long digestive tract compared with those of M. californica. Our results demonstrated that long-term organic amendment application significantly shapes earthworm gut microbial communities and results in greater decomposition potential of the earthworm gut. Furthermore, our study found that lower application rates of organic matter may be beneficial for earthworm gut microbial diversity and functional structures.